Nucleic acid detection or quantification method, chip and assay kit therefor, device for detecting or quantifying nucleic acid and program therefor

ABSTRACT

According to one embodiment, a method of quantifying a target nucleic acid containing a first sequence in a sample is provided. The method includes preparing a substrate on which a plurality of detection regions are arranged, forming a reaction field by placing, on the substrate, a reaction liquid containing a sample, a primer set, and an amplification enzyme, maintaining the reaction field in an isothermal amplification condition, detecting a detection signal for each of the detection regions, determining, for each of the plurality of detection regions, whether positive or negative and detecting or quantifying the target nucleic acid based on the number of positive and/or a rise time of each of the positive detection signal.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is based upon and claims the benefit of priority fromJapanese Patent Application No. 2017-118585, filed Jun. 16, 2017, theentire contents of which are incorporated herein by reference.

FIELD

Embodiments described herein relate generally to a nucleic aciddetection or quantification method, a chip and an assay kit therefor, adevice for detecting or quantifying nucleic acid and a programtherefore.

BACKGROUND

At present, with progress of genetic-testing technology, the nucleicacid testing is carried out in various scenes such as clinical diagnosisand criminal investigations. The target genes are detected or quantifiedby, for example, the real-time PCR method or LAMP method. The real-timePCR method is accompanied by the amplification of nucleic acid, andtherefore its sensitivity is high and the quantitative range is wide.The LAMP method can quantify or detect target genes without labeling theamplification product with a fluorochrome or the like. However, thequantifications or detections by these methods entail low accuracy inanalysis.

As a technology with higher accuracy in quantification or detection, thedigital PCR method has been introduced in practice. However, the digitalPCR method requires to adjusting the concentration of the reactionliquid appropriately and dispensing the liquid into a great number ofcontainers at an equal amount, which problematically involve complicatedoperations.

Under such circumstances, there is a demand for further development of aquantification or detection method which can detect nucleic acid simplyat high sensitivity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flowchart showing an example of a nucleic acid detection orquantification method in an embodiment.

FIG. 2 is a diagram showing an example of a substrate of the embodiment.

FIG. 3 is a diagram showing an example of a substrate of the embodiment.

FIG. 4 is an enlarged schematic diagram showing a flow channel of asubstrate of the embodiment.

FIG. 5 is a flowchart showing an example of a nucleic acid detection orquantification method of the embodiment.

FIG. 6 is a flowchart showing an example of a nucleic acid detection orquantification method of the embodiment.

FIG. 7 is a flowchart showing an example of a nucleic acid detection orquantification method of the embodiment.

FIG. 8 is a block diagram showing an example of a device for detectingor quantifying nucleic acid of the embodiment.

FIG. 9 is a flowchart showing an example of a nucleic acid detectingprocess by the device for detecting or quantifying nucleic acid of theembodiment.

FIG. 10 is a flowchart showing an example of processing of a nucleicacid quantification unit of the embodiment.

FIG. 11 is a graph showing experimental results in Example 2.

DETAILED DESCRIPTION

In general, according to one embodiment, a nucleic acid detection orquantification method is a method of quantifying a target nucleic acidcontaining a first sequence in a sample. The method comprises preparinga substrate on which a plurality of detection regions are arranged;forming one reaction field by placing, on the substrate, a reactionliquid containing a sample, a primer set for isothermally amplifying thefirst sequence to obtain an amplification product, and an amplificationenzyme; maintaining the reaction field in an isothermal amplificationcondition; detecting a detection signal varying with an increase of theamplification product in each of the plurality of detection regions;determining, for each of the plurality of detection regions, whether anamplification product exists in a vicinity (positive) or anamplification product does not exist (negative) based on the result ofthe detecting; and detecting or quantifying the target nucleic acidbased on the number of positive detection regions and/or a rise time ofthe detection signal in each of the positive detection regions.

Various embodiments will be described below with reference to theaccompanying drawings. Each figure is an exemplary diagram of anembodiment to aid understanding of the embodiment. The shapes,dimensions or ratios in the drawings may differ from those of the actualdevice, and may be appropriately changed in light of the subsequentexplanation and the known art.

FIG. 1 shows a brief flow of an example of the nucleic acid detection orquantification method according to an embodiment.

The nucleic acid detection or quantification method is a method ofquantifying a target nucleic acid containing the first sequence in asample. The method comprises: (S1) preparing a substrate on which aplurality of detection regions are arranged; (S2) forming one reactionfield by placing, on the substrate, a reaction liquid containing asample, a primer set for isothermally amplifying the first sequence toobtain an amplification product, and an amplification enzyme; (S3)maintaining the reaction field in an isothermal amplification condition;(S4) detecting a detection signal varying with an increase of theamplification product in each of the plurality of detection regions;(S5) determining, for each of the plurality of detection regions,whether an amplification product exists in a vicinity (positive) or anamplification product does not exist (negative) based on the result ofthe detecting; and (S6) detecting or quantifying a target nucleic acidbased on the number of positive detection regions and/or the rise timeof the detection signal in each of the positive detection regions.

Each processing step will be described in detail below.

In step (S1), a substrate on which a plurality of detection regions isarranged is prepared.

The substrate is a solid phase which supports one reaction field to beformed in step (S2) described later. As the substrate, for example,metal, resin, glass, silicon or the like can be used. The shape of thesubstrate as a whole may be, for example, plate-like, container shape,or a part of each of these.

An example of the substrate is shown in FIG. 2, part (a). In one surface2 of the substrate 1, a plurality of detection regions 3 are disposed.The detection regions 3 are those for detecting the detection signalfrom the reaction field formed on the one surface 2 of the substrate 1in step (S2) described later.

As a material of the detection regions 3, metal, resin, glass, siliconor the like can be used. The material of the detection regions 3 isselected according to the material of the substrate, the type of thedetection signal detected in step (S4), which will be described later.

The detection regions 3 are arranged in, for example, a regular array.Or the arrangement of the detection regions 3 may be random dispositionat a uniform density. It is preferable that the detection regions 3 bearranged uniformly on all over the region adjacent to the reaction fieldon the surface 2 of the substrate 1, formed in the step (S2), which willbe described later. With such a configuration, target nucleic acids aredetected or quantified more accurately.

When the detection regions 3 are arranged in a regular array, it ispreferable that intervals between adjacent pairs of detection regions,for example, arranged along a row direction and a column direction besubstantially equal to each other.

The size of each detection region 3 should just be set depending on thepredetermined size of the substrate 1 and the predetermined number ofthe detection regions 3, but, for example, the length is 0.001 to 10 mmand the width is 0.001 to 10 mm.

The shape of each the detection regions 3 should just be selectedaccording to the type of signal so as to be able to detect apredetermined signal to be detected, and, for example, it is circular,quadrangular or polygonal. The number of the detection regions 3arranged on a single substrate 1 should preferably be, for example, twoor more. Ten or more is preferable because in which case, the accuracyin detection and quantification further improves.

Further, it is preferable that the detection regions 3 be the same as anidentical to each other in area and shape. In particular, if thedetection regions vary from each other in area, the accuracy inquantification degrades. Therefore, the dispersion in area shouldpreferably be no more than 10%, or more preferably no more than 5%.

The detection regions 3 are arranged at an interval d1 along the rowdirection and an interval d2 along the column direction. That is, theintervals d1 and d2 are distances between two adjacent detection regionsarranged along the row direction and the column direction, respectively.In other words, they are each the closest distance between two detectionregions 3 adjacent to each other from an end of one detection region 3to a corresponding end of the other detection region 3. The intervals d1and d2 should preferably be no less than 0.1 mm. The intervals d1 and d2should more preferably be no less than 0.5 mm, and even more preferablyno less than 1 mm.

When the intervals d1 and d2 are 0.1 mm or more, such a phenomenon thata detection signal to be detected in a specific detection region isdetected also in a different detection region arranged near can besuppressed, and the accuracy in quantification can be improved. If noless than 0.5 mm, the phenomenon is further suppressed, thereby furtherimproving the accuracy in quantification. Especially, when 1 mm or more,even under such conditions that the target nucleic acid is short inlength and the reaction time is long condition, the phenomenon that adetection signal to be detected in a specific detection region isdetected also in a different detection region arranged near can beprevented, thereby further enhancing the accuracy in detection andquantification.

Here, the intervals d1 and d2 should preferably be no more than 10 mm.If the interval is excessively wide, the number of detection regionsformable on one substrate decreases. As a result, the accuracy inquantification degrades and a great quantity of sample is required toachieve a desired accuracy in quantification.

The substrate 1 may comprise sensors 4 corresponding respectively to thedetection regions 3. The sensors 4, which will be described later indetail, are, for example, electrodes, electrochemical sensors, opticalsensors, or turbidity sensors. When the sensors 4 are provided, all ofthose corresponding respectively to the detection regions 3 on onesubstrate 1 should preferably be of the same kind.

The detection regions 3 may be each formed from a light-transmittingmaterial. That is, the portions of the substrate 1, which correspond tothe detection region 3, may be formed from a light-transmittingmaterial. In that case, the detection signal can be detected from thedetection regions with, for example, a sensor separated from thesubstrate 1.

In the substrate 1 with the above-described structure, one space isformed on the surface 2. That is, the space on the surface 2 is notdivided for a plurality of detection regions 3. With this structure,when the reaction liquid is brought onto the surface 2 in step (S2),which will be described later, the reaction mixture is placed in such astate that it can flow to any location on the surface 2. As a result,one reaction field is formed. Therefore, in the substrate 1, it ispossible to bring the reaction liquid onto all the detection regions 3by one operation. Further, the volumes of the spaces on these respectivedetection regions 3 are constant.

Part (b) of FIG. 2 shows a further example of the substrate. In thisexample, a surface 12 of a substrate 11 comprises a flow channel 13formed from a meandering groove. It is preferable that detection regions15 be arranged by one or a plurality of rows along with the flow channel13 in a bottom portion 14 of the flow channel 13. A width w1 of the flowchannel 13 may be determined according to, for example, the size of thesubstrate 11, and preferably, for example, 0.01 to 50 mm. A depth of theflow channel should preferably be, for example, 0.01 to 10 mm. The sizeof each detection region 15 may be selected depending on the size of adesired substrate 11, the number of desired detection regions 15, thewidth of the flow channel 13, or the like, but, for example, the lengthis 0.001 to 10 mm, and the width is 0.001 to 10 mm, etc.

The distance of two detection regions 15 adjacent to each other, thatis, a closest interval d3 between the two regions from an end of onedetection region 15 to an end of the other detection region 15 shouldpreferably be no less than 0.1 mm, or more preferably no less than 0.5mm or more, or even more preferably no less than 1 mm.

If the interval d3 is no less than 0.1 mm, such a phenomenon that adetection signal to be detected in a specific detection region isdetected also in a different detection region arranged near does noteasily occur, and the accuracy in quantification can be improved. If notless than 5 mm, the phenomenon is further suppressed and the accuracy inquantification further improves. Especially, if no more than 1 mm, evenunder conditions that the length of target nucleic acid is short and thereaction time is long, the phenomenon that a detection signal to bedetected in a specific detection region is detected also in a differentdetection region arranged near can be prevented; therefore the accuracyin detection and quantification is further enhanced.

The number of detection regions 15 arranged in one substrate 11 shouldpreferably be, for example, two or more. Ten or more is more preferably,the accuracy in detection is further enhanced.

The shape of the flow channel 13 may be one meandering groove such asthat shown in part (b) of FIG. 2, or may be some other shape. Forexample, it may be a comb-like shape as the flow channel 23 shown inpart (a) of FIG. 3, or a square spiral shape as the flow channel 33shown in part (b) of FIG. 3, or a circular spiral shape as the flowchannel 43 shown in part (c) of FIG. 3.

The corners of the flow channels 13, 23 and 33 should preferably bechamfered. With the chamfered corners, it is possible to suppress theproduction of bubbles while bringing the reaction liquid into the flowchannel in step (S2), which will be described later, which may causeblocking of the amplification reaction.

The substrates 11, 21, 31 and 41 may respectively comprise sensors 16,26, 36 and 46 similar to the above-described sensor 4, in the detectionregions 15, 25, 35 and 45. Or, the detection regions 15, 25, 35 or 45may be formed from a light-transmitting member as described above.

Next, in step (S2), the reaction liquid is placed on the surface of thesubstrate, where a plurality of detection regions exists, so as to forma reaction field. A “reaction field” is a region where an amplificationreaction occurs, and the region is defined by the reaction liquid. Inother words, a reaction field is a region where a reaction liquidexists.

The reaction liquid contains a sample, a primer set and an amplificationenzyme.

The sample is a substance to be examined as to the presence/absence orquantity of a target nucleic acid. In other words, a sample is an objectto be analyzed in the nucleic acid detection or quantification method ofthe embodiment. For example, the sample may be bio-materials containingblood, serum, leukocyte, urine, feces, sweat, saliva, oral mucosa,expectoration, lymph, spinal fluid, lacrimal fluid, mother milk,amniotic fluid, semen, tissue, biopsy and culture cells, environmentalmaterials collected from the environment, artificial nucleic acids orthe like, or mixtures of those. Further, a preparation formulated usingany of above materials may as well be used as the sample. For example, apretreatment may be carried out on any of above materials to be used asa sample in the embodiment. The pretreatment may be any conventionalmeans known by itself, such as a fragment, homogenization or extraction,for example. For example, any of above materials may be collected froman organism or environment, and formulated into a condition suitable forthe nucleic acid detection. For example, a liquid containing a nucleicacid component which is obtained by extracting a nucleic acid from anyof the above materials by any means can be used as the sample.

Target nucleic acid is nucleic acid which should be detected orquantified in the nucleic acid detection or quantification method of theembodiment. The target nucleic acid includes the first sequence. Thefirst sequence is a sequence used as the index of the existence of thetarget nucleic acid, and a sequence to be amplified in the nucleic aciddetection or quantification method of the embodiment so as to detect orquantify the target nucleic acid. The first sequence is a sequenceselected from the sequence covering the full length of the targetnucleic acid, and should preferably be, for example, a sequence specificto the target nucleic acid. The target nucleic acid is a single strandnucleic acid. The state of the target nucleic acid in the sample may bea single strand or a double strand formed from a target nucleic acid anda nucleic acid chain complementary to the target nucleic acid. Thelength of the target sequence may be, for example, 50 to 500 bases, andpreferably, 100 to 300 bases.

The length of the first sequence may be, for example, 3 to 10 bases, 10to 20 bases, 20 to 30 bases, 30 to 40 bases, 40 to 50 bases and 50 to 60bases, 60 to 70 bases, 70 to 80 bases, 80 to 90 bases or 90 to 100bases, and preferably, 10 to 50 bases.

The primer set is that for isothermal amplification for amplifying theabove-described first sequence to obtain an amplification product. Basedon the kind of the amplification method used for the nucleic aciddetection or quantification method, the sequence of each primercontained in the primer set should just be designed and/or selected soas to amplify the first sequence. The amplification method used for thenucleic acid detection or quantification method is an isothermalamplification method. The employable amplification method may be, forexample, LAMP, RT-LAMP, SDA, NASBA, RCA, LCR, TMA, SmartAmp (registeredtrademark) or ICAN (registered trademark). For example, the primer setcontains a first primer complementary to one terminal of the firstsequence and a second primer homologous to the other terminal of thefirst sequence. With these primers, a range to be amplified on thetarget nucleic acid is specified.

In the case where the primer set is that for LAMP, one primer set maycontain an FIP primer as the first primer and a BIP primer as the secondprimer. The primer set for LAMP may further contain an F3 primer, B3primer and LP primer, that is, LF primers and/or LB primers.

For example, when the target nucleic acid in a sample is asingle-stranded DNA, a complementary strand is formed by the primer set,and further the amplification reaction advances using it as a template.

Moreover, when the target nucleic acid is RNA, a reverse transcriptionreaction is carried out and the reverse transcription product issubjected to the amplification reaction.

The amplification enzyme may be selected based on the kind of each ofthe target nucleic acid, the isothermal amplification method employedand the primer set, and the presence/absence of a reverse transcriptionreaction, etc. The amplification enzyme may be DNA-polymerase,RNA-polymerase, or the like for example. The DNA polymerase shouldpreferably be, for example, Bst, Bst2.0, Bst3.0, GspSSD, GspM, Tin, Bsm,Csa, 96-7, phi29, Omini-Amp (registered trademark), Aac, BcaBEST(registered trademark), Displace Ace (registered trademark), SD, StrandDisplace (registered trademark), TOPOTAQ, Isotherm2G, Taq or acombination of any of these. Use of Bst, GspSSD or Tin is morepreferable because they enhance the sensitivity in the detection andquantification. In addition to the amplification enzyme, any reversetranscriptase may be further employed.

The reaction liquid may further contain magnesium in addition to theabove-described ingredients. The concentration of magnesium in thereaction liquid should just be selected based on the kind of thedetection signal, but should be, for example, no more than 30 mM, andmore preferably, 4 mM to 10 mM. With this concentration, theamplification reaction is promoted and also various sequences of a widerange, which do not depend on the sequence, can be amplifiedefficiently. Thus, various types of sequences can be detectedefficiently.

The reaction liquid may contain some other ingredients required for theamplification reaction in addition to the above-described ingredients.Such ingredients may be, for example, a marker substance which producinga signal according to an increase in amplification produce, a salt, asubstrate such as deoxynucleoside triphosphoric acid (dNTP), which isrequired to form a new polynucleotide chain whose origin of replicationis the primer, a thickener as a reaction reagent, a buffer for pHadjustment, a surfactant, ion for enhancing the annealing specificityand/or ion which gives rise to a cofactor of the amplification enzyme,etc. When performing a reverse transcription simultaneously, thereaction liquid may further contain a reverse transcriptase and asubstrate required therefor, and the like.

The marker substance is a substance producing the detection signal whichchanges with the increase in an amplification product. For example, themarker substance is a substance which increases or decreases theproduction of detection signals when an amplification product exists ascompared with the case where the amplification product does not exist.Or, for example, it is a substance which increases or decreases thequantity of the detection signal produced from there according to theamount of the amplification product presence. For example, the markersubstance is a substance producing an electric signal or an opticalsignal, or the like, as will be described later in detail. The reactionliquid may not necessarily contain a marker substance. In that case, thesignal correlated to the turbidity of the reaction liquid may be used asthe detection signal to quantify or detect the target nucleic acid.

The salt may be any of well-known salt used, for example, to maintain anappropriate amplification environment in the nucleic acid amplificationreaction. Maintaining an appropriate amplification environment in thenucleic acid amplification reaction means that, for example, theamplification enzyme maintains its tertiary structure so as to optimizethe nucleic acid amplification activity. The salt is potassium chloride,for example. The concentration of the salt in the reaction liquid shouldpreferably be, for example, 5 to 300 mol/L.

The reaction liquid described above is placed on the surface of thesubstrate, where the detection regions are present, to form one reactionfield. The one reaction field is a reaction field formed in onecontinuous region. For example, one reaction field is formed by bringinga reaction liquid on the surface of any of the above-describedsubstrates, where the detection regions are present.

For example, when using the substrate 1 illustrated in part (a) of FIG.2, a reaction field is formed by bringing the reaction liquid on thesurface 2 of the substrate 1. When using the substrate of illustrated inpart (b) of FIG. 2, a reaction field is formed by bringing the reactionliquid into the flow channel 13. In order to bring the reaction liquidto the flow channel 13, for example, the reaction liquid is injectedfrom a liquid inlet 13 a, and the air in the flow channel 13 isextracted from a discharge liquid outlet 13 b. When using the substrateillustrated in part (b) of FIG. 3, the reaction liquid can be broughtinto the flow channel 23 by, for example, injecting the reaction liquidfrom a liquid inlet 23 a and extracting the air from the dischargeliquid outlets 23 b to 23 f. When using the substrate illustrated inpart (b) of FIG. 3, the reaction liquid can be brought into the flowchannel 33 by, for example, injecting the reaction liquid from a liquidinlet 33 a and extracting the air from the discharge liquid outlet 33 b.When using the substrate illustrated in part (c) of FIG. 3, the reactionliquid can be brought into the flow channel 43 by, for example,injecting the reaction liquid from a liquid inlet 43 a and extractingthe air from the liquid discharge outlet 43 b.

The ingredients of the reaction liquid should just be each contained inthe reaction liquid which forms the reaction field. Therefore, forexample, the ingredients of the reaction liquid may be each contained inthe reaction liquid before the reaction liquid is brought onto thesubstrate. Alternatively, the ingredients of the reaction liquid may beprepared separately from each other and may be brought into the reactionliquid at the same time as, before or after the reaction liquid isbrought onto the substrate. Or before the reaction liquid is broughtonto the substrate, some of the ingredients may be releasablyimmobilized to a solid phase or the like, which is a surface in contactwith the reaction field, and brought into the reaction liquid by beingreleased into the reaction liquid when the reaction liquid is broughtthereto.

In step (S3), the reaction filed is maintained in an isothermalamplification condition.

The isothermal amplification condition is selected based on, forexample, the kind of the isothermal amplification method employed, thekind of the primer set, the kind of the target nucleic acid, and/or thekind of the amplification enzyme, etc. Maintaining the isothermalamplification reaction condition is, for example, maintaining thetemperature of the reaction field at 25° C. to 70° C. It is morepreferable to maintain the temperature at 55° C. to 65° C. Theisothermal amplification reaction condition should preferably be a LAMPamplification reaction condition.

When target nucleic acid exists in a sample, the amplification reactionoccurs by maintaining the reaction field under an isothermalamplification reaction condition, and the first sequence thereof isamplified, and an amplification product is produced.

The amplification product is produced in the position where the targetnucleic acid exists on the surface of the substrate, where the detectionregion is disposed, and it increases and remains in its vicinity. Forexample, the amplification product amplified from one target nucleicacid molecule remains in a range of 0.001 to 1 mm from the positionwhere the target nucleic acid initially existed. On the contrary, theamplification product hardly exists in the positions other than wherethe target nucleic acid exists or its vicinity on the surface.

In step (S4), the detection signal is detected in each of the detectionregions.

The detection signal is a signal to be detected, which varies with theincrease in the amplification product. Such a signal is, for example, anelectric signal or optical signal produced from the marker substancewhich exists in the reaction field or a signal correlated to theturbidity of the reaction liquid, as will be described later in detail.

The detection signal is detected in a detection region. The detectionsignal may be detected by the above-described sensor provided in thedetection region. Or the detection signal may be detected from adetection region by a sensor or the like, separated from the substrate.The detection signal is detected in all the detection regions arrangedon the substrate.

The detection is performed at, for example, the end point of theamplification reaction, or may be performed sequentially. The sequentialdetection may be continuous, or intermittent, in which the detection iscarried out a plurality of times at a predetermined time interval. Forexample, the continuous detection may be monitoring of the detectionsignal. It may detect the rise time of the detection signal.

In step (S5), it is determined whether an amplification product exists(positive) or does not exist (negative) in the vicinity of each of thedetection regions.

When the detection of step (S4) is performed in the end point of anamplification reaction, the detection signal from a detection regiondisposed in the position on the substrate, where the amplificationproduct is produced and increased increases or decreases as compared tothe detection signal before the amplification reaction, or the detectionsignal from a detection region where the target nucleic acid does notexists therearound. Therefore, a detection region where the detectionsignal increases or decreases as compared to that before theamplification or other detection region can be determined as that “anamplification product exists in its vicinity (positive)”. On the otherhand, a detection region which is not so can determined as that “anamplification product does not exist in its vicinity (negative)”.

The term “vicinity” is defined as within a range where the existence ofan amplification product can detected with a detection means employed,such as a sensor. An example of the range in which the existence of anamplification product can be detected is shown in parts (a) and (b) ofFIG. 4. Part (a) of FIG. 4 is an enlarged plan view of a portion Bencircled in part (b) of FIG. 2. Part (b) of FIG. 4 is a cross sectiontaken along line B′-B′ in part (a) of FIG. 4, part (a). A range R inwhich the existence of an amplification product can be detected is aregion in the reaction field in the flow channel 13. The region is thatdefined as the distance from an edge of a detection region 15 isspecified by a value, and is approximately hemispherical. The distancevaries depending on the detection means, but it is, for example, 0 to 10mm.

Based on the determination made above, the number of positive detectionregions and the number of negative detection regions in one reactionfield can be obtained.

When the detection of step (S4) is carried out with time, the detectionsignal is produced from a detection region on the substrate, located ina position where the amplification product is produced and increased.This detection signal has a rise time, that is, the time required forthe size of the detection signal to exceed a predetermined threshold,shorter as compared to that of the detection signal from a detectionregion around where a target nucleic acid does not exist. Therefore, adetection region where the rise of an increase of a detection signal isobserved in a shorter time as compared to other detection regions isdetermined as that “an amplification product exists in its vicinity(positive)” and a detection region which is not so is determined as that“an amplification product does not exist in the vicinity (negative)”.Based on this, the number of positive detection regions and the numberof negative detection regions in one reaction field can be determined.

In step (S6), the target nucleic acid is detected or quantified from thenumber of positive detection regions obtained in step (S5) and/or therise time of the detection signal in each of the positive detectionregions.

For example, when the number of positive detection regions is 0, it isdetermined that any target nucleic acid does not exist in the sample.

When both positive and negative detection regions exist, the quantity ofthe target nucleic acid present in the sample can be calculated from thenumber of positive detection regions. The calculation may be based on,for example, a statistical procedure. As the statistical procedure, amost probable number (MPN) method can be used.

The most probable number method is a procedure of acquiring the maximumlikelihood estimated value (most probable number: MPN) of the quantityof a target nucleic acid in a sample by the following formula 1.MPN=(Σ_(gi))/(Σ_(tjmj)Σ(tj−gj)_(mj))½  Formula 1

where;

Σ_(gi) is a sum of the values of the positive detection regions;

Σ_(tjmj) is a sum of values of (number of detection regions×dilutionrate); and

Σ(tj−gj)_(mj) is a sum of values of (number of negatives×dilution rate).

When the reaction liquid is not diluted, the most probable number (MPN)can be obtained by the following Formula 2.MPN=1/m×2.303×log((number of detection regions)/(number ofnegatives))  Formula 2

where m is a reaction volume per detection region.

The reaction volume per detection region can be obtained by calculatingout m, which is obtained by, for example, applying to Formula 2, astandard sample nucleic acid of a known concentration is amplified on asubstrate to be used, thus detecting detection signals, and the numberof positives obtained by detecting the detection signals, theconcentration of the standard sample nucleic acid and the number ofdetection regions on the substrate. With this method, it is possible todetect, for example, 1 to 10⁴ copies/mL of target nucleic acid containedin the sample.

Incidentally, when a great number of target nucleic acids exist in asample, the target nucleic acids existing in the vicinity of everydetection region may be detected as positive. Here, as the quantity oftarget nucleic acid existing in the sample is greater, the rise in theincrease of the detection signal is observed in a shorter time in allthe detection regions. In that case, the target nucleic acid can bedetected or quantified by, for example, the following manner. Aplurality of different standard sample nucleic acids whose quantities ofnucleic acid present are already known are used to prepare a calibrationcurve of the rise time of the detection signal with respect to thequantity of nucleic acid present, and the calibration curve is comparedwith the measurement result of the rise time in the target nucleic acid.Thus, the quantity of the target nucleic acid present in a sample can becalculated. With this method, it is possible to detect, for example, 10⁴to 10⁹ copies/mL of target nucleic acid contained in the sample.

With step (S6) described above, the target nucleic acid in a sample canbe detected or quantified.

According to the nucleic acid detection or quantification methoddescribed above, there is no need to dilute a sample or divide thereaction liquid, but with such a simple process of forming one reactionfield, that is, the reaction liquid is brought onto one continuousregion on a substrate, the target nucleic acid can be detected at higheraccuracy. For example, according to the method of the embodiment, it ispossible to detect, for example, 1 to 10⁹ copies/mL of target nucleicacid contained in the sample.

The above-described procedure can be realized because the amplificationmethod is of the isothermal type. To explain, if an amplification methodsuch as PCR method carried out in temperature variation, are used, theconvection of the reaction liquid occurs during the reaction, whicheventually causes diffusion of the amplification product, and theamplification product may move even to the position of a detectionregion where the target nucleic acid does not initially exist.Therefore, the presence/absence and/or the quantity of the amplificationproduct existing in the vicinity of a predetermined detection region cannot be correctly detected. However, with the method of the embodiment,which adopts the isothermal amplification, the amplification productdoes not move but remains at the position where the target nucleic acidinitially existed. In this manner, the detection region where the targetnucleic acid exists can be identified accurately, and the quantity ofthe amplification product is accurately reflected in the detectionsignal. Therefore, it is possible to accurately quantify and detect thetarget nucleic acid.

For example, with a substrate comprising any of the flow channelsdescribed above, it becomes even harder for the reaction liquid to move,and therefore the target nucleic acid can be quantified or detected evenmore accurately.

Moreover, according to the nucleic acid detection or quantificationmethod of the embodiment, the amplification reaction is carried out on asubstrate comprising a plurality of detection regions located atpredetermined positions, and the detection signals are obtained from allthe detection regions. With this structure, regardless of what positionsof the reaction field, the target nucleic acid exists, the informationcovering the entire reaction field can be obtained without bias.Therefore, it is possible to estimate more accurately the positionswhere the target nucleic acid exists. As a result, the target nucleicacid can be detected and quantified at higher accuracy. Here, forexample, even in the case where the target nucleic acids of samplesobtained from a plurality of objects are to be detected or quantified,if substrates of the same structure are used according to the nucleicacid detection or quantification method of the embodiment, the resultscan be obtained at the same accuracy regardless of the samples. Thus,even more highly reliable detection and quantification can be carriedout. In the case where the results obtained are compared each otheramong the samples as well, an even more highly reliable comparisonresults can be obtained.

Examples of the nucleic acid detection or quantification methoddescribed above, in which an electric signal, an optical signal, and asignal correlated to turbidity are used, respectively, will be describedin detail.

The Nucleic Acid Detection or Quantification Method Using ElectricSignal.

FIG. 5 shows a flowchart schematically illustrating the nucleic aciddetection or quantification method using electric signal.

The nucleic acid detection or quantification method comprises: (S101)preparing a substrate on which a plurality of detection regions eachcomprising an electrode are arranged; (S102) forming one reaction fieldby placing, on the substrate, a reaction liquid containing a sample, aprimer set for isothermally amplifying the first sequence to obtain anamplification product, an amplification enzyme and a first markersubstance producing an electric signal varying with the increase of theamplification product; (S103) maintaining the reaction field in anisothermal amplification condition; (S104) detecting an electric signalwith each of the electrodes; (S105) determining whether an amplificationproduct exists in its vicinity (positive) or an amplification productdoes not exist in ins vicinity (negative) for each of a plurality ofdetection regions based on the result of the detection; and (S106)detecting or quantifying the target nucleic acid from the number ofpositive detection regions and/or the rise time of the electric signalsin each of the positive detection regions.

In step (S101), a substrate on which a plurality of detection regionseach comprising an electrode are arranged, is prepared.

As the substrate, for example, any one of those shown in FIGS. 2 and 3,which comprise electrodes corresponding to a plurality of detectionregions, can be used. The electrodes can be obtained, for example, byforming metal patterns of a predetermined shape such as a dot on each ofthe detection regions on the substrate. The metal patterns can beformed, for example, by using a photolithographic method. Such a methodis preferable because thereby more electrodes can be formed on thesubstrate. As the metal, for example, gold is preferable because of itshigh sensitivity.

It is preferable to form, for example, two or more detection regions,that is, electrodes, on one substrate. It is even more preferable toform ten or more, because in which case the accuracy is furtherimproved.

The electrodes are each disposed so as to be able to detect the electricsignal from the first marker substance existing on the reaction field.In other words, the electrodes are each arranged so as to be at leastpartially brought into contact with the reaction liquid when thereaction liquid is brought onto the substrate to form the reactionfield.

The substrates each may further comprise a pad. The pad is electricallyconnected to a respective electrode, from which the data on the electricsignal obtained with the electrode can be extracted. Further, thesubstrate may further comprise a reference electrode and a counterelectrode.

In step (S102), the reaction liquid is placed on the surface of thesubstrate, where a plurality of detection regions exists, and thus areaction field is formed.

The reaction liquid contains a sample, a primer set, an amplificationenzyme and a first marker substance. The sample, primer set andamplification enzyme may be any one of those described above.

The first marker substance is a substance producing an electric signalupon the increase of the amplification product. For example, the firstmarker substance is, for example, an oxidizer whose oxidation-reductionpotential can be an electric signal.

Examples of the first marker substance are ferricyanide ion,ferrocyanide ion, an iron complex ion, a ruthenium complex ion and acobalt complex ion. These marker substances can be each obtained bydissolving potassium ferricyanide, potassium ferrocyanide, an ironcomplex, a ruthenium complex or a cobalt complex into a reaction liquid.The concentrations in those reaction liquids may be, for example, 10 μMto 100 mM, or about 1 mM.

For example, when ferricyanide ion (Fe(CN)₆ ⁴—) is used as the firstmarker substance, electrons are emitted by the oxidation reaction ofFe(CN)₆ ⁴— into Fe(CN)₆ ³—. These electrons repel the amplificationproduct having negative charge and move away from the amplificationproduct. Therefore, in an electrode around which an amplificationproduct exists, the current (electric signal) detected decreases as theamplification product increases.

The first marker substance may be used in combination with anothermarker substance. When an electrochemically active substance havingnegative or positive charge and for example, a nucleic acid probelabeled with ferrocene are used in combination in a reaction field,ferrocene serves as a mediator to amplify the electric signal, and thusthe sensitivity is further improved.

Or the first marker substance may be a redox probe. The redox probe is asubstance which has an oxidation reduction potential of, for example,−0.5V to 0.5V, and it electrostatically binds to the amplificationproduct in the reaction liquid. By applying voltage to the electrode,the redox probe bound to the amplification product is oxidized orreduced, and electrons are emitted by the reaction. Therefore, forexample, in the electrode disposed in the position where theamplification product exists, the current (electric signal) detectedincreases as the amplification product increases, or the peak potentialof the oxidation-reduction potential detected is shifted in a negativedirection.

In the electrode around which an amplification product exists, whetherthe current (electric signal) detected increases as the amplificationproduct increases or the peak potential of the oxidation-reductionpotential detected is shifted in the negative direction can be adjusted,for example, by changing the magnesium concentration in the reactionliquid. For example, when the magnesium concentration is 4 mM to 30 mM,the current to be detected can increase as the amplification productincreases. Or as the amplification product increases, the peak potentialof the oxidation reduction potential to be detected can be shifted inthe negative direction. Therefore, it is possible to carry out even ahigher-accuracy measurement by further detecting the peak potential ofthe oxidation-reduction potential in addition to the electric signal.

The redox probe is a metal complex, for example. The metal complex to beused as the redox probe may contain, for example, ruthenium (Ru),rhodium (Rh), platinum (Pt), cobalt (Co), chromium (Cr), cadmium (Cd),nickel (Ni), zinc (Zn), copper (Cu), osmium (Os), iron (Fe), or silver(Ag) as a central metal. The metal complex may be, for example, aminecomplex, cyano complex, halogen complex, hydroxy complex,cyclopentadienyl complex, phenanthroline complex or bipyridine complex.Further, redox probes such as methylene blue, Nile blue and crystalviolet, can also be used.

For example, the first marker substance is ruthenium hexaamine (RuHex).Here, when an amplification product exists, RuHex³⁺ bound to theamplification product applies voltage to the electrode to be reduced toRuHex²⁺, thereby emitting an electron. As the electron flows into theelectrode, the amplification product can be detected.

The concentration of the redox probe in the reaction liquid is, forexample, 0.1 μM to 100 mM, but preferably, 25 μM to 3 mM, and morepreferably, 1 mM in which case, the detection sensitivity of the nucleicacid can be improved. If excessively less, it cannot sufficiently bindto the amplification product and the sensitivity may be undesirablydegraded. On the other hand, if excessively high, the amplificationreaction may be blocked. Especially when the first marker substance isruthenium hexaamine (RuHex), it is preferable that RuHex be contained inthe reaction liquid in a range of 25 μM to 3 mM.

The reaction mixture may contain magnesium at a desired concentration tobe selected based on the kind of the first marker substance or the kindof detection signal or the like as described above.

The reaction mixture may contain a salt, a substrate such asdeoxynucleoside triphosphoric acid (dNTP), which is required to form anew polynucleotide using the primer as an origin of replication, athickener as a reaction reagent, a buffer for pH adjustment, asurfactant, ion for enhancing the annealing specificity and/or ion whichgives rise to a cofactor of the amplification enzyme, etc. When carryingout reverse transcription simultaneously, the reaction liquid maycontain reverse transcriptase and a desired ingredient required for theamplification reactions, such as a substrate required therefor.

The reaction liquid described above is placed on the surface of thesubstrate, where the detection regions are disposed, to form onereaction field. The reaction field can be formed by a method similar toany of those described above.

In step (S103), the reaction field is maintained in the isothermalamplification condition. This step can be carried out, for example, by aprocedure similar to that of step (S3) described above.

In step (8104), the electric signal is detected with an electrode in aplurality of detection regions.

The electric signal is obtained from the first marker substance. Theelectric signal may be, for example, a current value, a potential value,a capacitance value, an impedance value or the like. The signal isdetected by each electrode provided in the detection regions. Forexample, a plurality of kinds of electric signals such as a currentvalue and a potential value may be measured. The detection may becarried out, for example, by a procedure similar to that described instep (S4) described above, for example, may be carried out at the endpoint of the reaction or sequentially during the reaction.

In step (S105), it is determined whether an amplification product exists(positive) or does not exist (negative) in the vicinity of each of thedetection regions based on the result of the detection. This step can becarried out, for example, by a procedure similar to that of step (S5)described above. In this embodiment, the “vicinity” is defined, forexample, as a region on the reaction field, located within such adistance from an edge of the electrode provided in the respectivedetection region is in a range of 0 to 10 mm.

In step (S106), the target nucleic acid is quantified from the number ofpositive detection regions, and/or the rise time of the electricsignals. This step can be carried out, for example, by the sameprocedure as that of step (S6) described above.

According to the method described above, the target nucleic acid can bequantified simply at high sensitivity.

As an alternative embodiment which adopts the nucleic acid detection orquantification method using an electric signal, an electrochemicalsensor may be employed in place of the electrode. Here, theelectrochemical sensor should just be a well-known electrochemicalsensor which can detect an electric signal from the above-describedfirst marker substance.

The Nucleic Acid Detection or Quantification Method Using Optical Signal

FIG. 6 is a flowchart schematically illustrating a nucleic aciddetection or quantification method using an optical signal.

The nucleic acid detection or quantification method comprises: (S201)preparing a substrate on which a plurality of detection regions eachcomprising an optical sensor are arranged; (S202) forming one reactionfield by placing, on the substrate, a reaction liquid containing asample, a primer set for isothermally amplifying the first sequence toobtain an amplification product, an amplification enzyme and a secondmarker substance producing an optical signal varying with the increaseof the amplification product; (S203) maintaining the reaction field inan isothermal amplification condition; (S204) detecting an opticalsignal with each of the optical sensors; (S205) determining whether anamplification product exists in its vicinity (positive) or anamplification product does not exist in ins vicinity (negative) for eachof a plurality of detection regions based on the result of thedetection; and (S206) detecting or quantifying the target nucleic acidfrom the number of positive detection regions and/or the rise time ofthe optical signals in each of the positive detection regions.

In step (S201), a substrate on which a plurality of detection regionseach comprising an optical sensor are arranged, is prepared.

As the substrate, for example, any one of those shown in FIGS. 2 and 3,which comprise optical sensors corresponding to a plurality of detectionregions, can be used. The optical sensors are each disposed so as to beable to detect the optical signal from the second marker substanceexisting on the reaction field. The optical sensor should just be anyconventionally known sensor which can detect fluorescence, lightemission or the like. For example, the optical sensor is an element orthe like, which detect an optical signal such as fluorescence orluminescence and convert it into an electric signal.

Or as the substrate, for example, any of those shown in FIGS. 2 and 3,in which each of the detection regions is formed from alight-transmitting material can be employed. The light-transmittingmaterial is, for example, resin. In this case, for example, signals canbe detected by an optical sensor separated from the substrate. Or theoptical signals of each detection region may be acquired by obtainingthe images of all the detection regions may be acquired, and analyzingcolor, brightness and/or the like of a region corresponding to therespective detection region of the image.

In step (S202), the reaction field is formed on the substrate with thereaction liquid.

The reaction liquid contains a sample, a primer set, amplificationenzyme and the second marker substance.

The sample, primer set and amplification enzyme to be used may be any ofthose described above.

The second marker substance is a substance producing an optical signalupon the increase of the amplification product. The optical signal is,for example, light having a specific wavelength such as fluorescence orlight emission. The second marker substance is, for example, a substancewhich emits fluorescence, and when binds to an amplification product,increase the fluorescent value in its detection region. Usable examplesof the second marker substance are SYBRGreen, EvaGreen, SYTO, Berberine,Calcein, and HNB. Such a marker substance should preferably be containedin the reaction liquid at a concentration of 0.001 μM to 10 mM.

In addition to these ingredients, the reaction liquid may furthercontain magnesium, a salt, a substrate such as deoxynucleosidetriphosphoric acid (dNTP), which is required to form a newpolynucleotide using the primer as an origin of replication, a thickeneras a reaction reagent, a buffer for pH adjustment, a surfactant, ion forenhancing the annealing specificity and/or ion which gives rise to acofactor of the amplification enzyme, etc. When carrying out reversetranscription simultaneously, the reaction liquid may contain reversetranscriptase and a predetermined ingredient required for theamplification reactions, such as a substrate required therefor.

The reaction liquid described above is placed on the surface of thesubstrate, where the detection regions are disposed, to form onereaction field. The reaction field can be formed by any of the methodsdescribed above.

In step (S203), the reaction field is maintained in the isothermalamplification condition. This step can be carried out, for example, by aprocedure similar to that of step (S3) described above.

In step (S204), the optical signal is detected by the respective opticalsensors in a plurality of detection regions. The optical signal is theabove-described optical signal produced from the second markersubstance. The detection may be carried out, for example, by a proceduresimilar to the method described in step (S4), for example, may becarried out at the end point of the reaction or sequentially during thereaction.

In step (S205), it is determined whether an amplification product exists(positive) or does not exist (negative) in the vicinity of each of thedetection regions based on the result of the detection.

This step can be carried out, for example, by a procedure similar tothat of step (S5) described above. In this embodiment, the “vicinity” isdefined, for example, as a region on the reaction field, located withinsuch a distance from an edge of the optical sensor provided in therespective detection region is in a range of 0 to 10 mm.

In step (S206), the target nucleic acid is quantified from the number ofpositive detection regions, and/or the rise time of the optical signals.This step can be carried out, for example, by the same procedure as thatof step (S6) described above.

According to the method described above, the target nucleic acid can bequantified simply at high sensitivity.

The Nucleic Acid Detection or Quantification Method Using SignalCorrelated to Turbidity

FIG. 7 is a flowchart schematically illustrating a nucleic aciddetection or quantification method using a signal correlated toturbidity as the detection signal.

The method comprises: (S301) preparing a substrate on which a pluralityof detection regions each comprising a turbidity sensor are arranged;(S302) forming one reaction field by placing, on the substrate, areaction liquid containing a sample, a primer set for isothermallyamplifying the first sequence to obtain an amplification product, and anamplification enzyme; (S303) maintaining the reaction field in anisothermal amplification condition; (S304) detecting a detection signalcorrelated to turbidity with each of the turbidity sensors; (S305)determining whether an amplification product exists in a vicinity(positive) or an amplification product does not exist (negative) foreach of the plurality of detection regions, based on the result of thedetecting; and (S306) detecting or quantifying a target nucleic acidbased on the number of positive detection regions and/or the rise timeof the signal correlated to the turbidity in each of the positivedetection regions.

In step (S301), a substrate on which a plurality of detection regionseach comprising a turbidity sensor are arranged, is prepared.

As the substrate, for example, any one of those shown in FIGS. 2 and 3,which comprise turbidity sensors respectively in the detection regions,can be used. The turbidity sensors should just be of any conventionallyknown type can detect a signal correlated to turbidity. The signalcorrelated to turbidity is a signal correlated to the turbidity of areaction liquid, which can estimate the turbidity, that is, for example,the color of the reaction liquid, the intensity of transmitted light orintensity of dispersion light when light is applied to the reactionliquid, etc. For example, the turbidity sensors are each an elementwhich detects signal correlated to turbidity and convert it into anelectric signal. The turbidity sensors are arranged so to be able todetect the respective signals.

As the substrate, any one of those illustrated in FIGS. 2 and 3, inwhich, for example, a plurality of detection regions are each formedfrom a light-transmitting material. The light-transmitting material is,for example, a resin. With this structure, the signals correlated toturbidity can be detected, for example, by turbidity sensors separatedfrom the substrate. Or the signals correlated to the turbidity of thesedetection regions may be acquired by obtaining the images of thedetection regions and analyzing the color, brightness, etc., of a regionequivalent to the detection region of each image.

In step (S302), the reaction field is formed on the substrate with thereaction liquid.

The reaction liquid contains a sample, a primer set and amplificationenzyme.

The sample, primer set and amplification enzyme may be any one of thosedescribed above.

In addition to these ingredients, the reaction liquid may furthercontain magnesium, a salt, a substrate such as deoxynucleosidetriphosphoric acid (dNTP), which is required to form a newpolynucleotide using the primer as an origin of replication, a thickeneras a reaction reagent, a buffer for pH adjustment, a surfactant, ion forenhancing the annealing specificity and/or ion which gives rise to acofactor of the amplification enzyme, etc. When carrying out reversetranscription simultaneously, the reaction liquid may contain a reversetranscriptase and a predetermined ingredient required for theamplification reactions, such as a substrate required therefor.

The reaction liquid described above is placed on the surface of thesubstrate, where the detection regions are disposed, to form onereaction field. The reaction field can be formed by any of the methodsdescribed above.

In step (S303), the reaction field is maintained in the isothermalamplification condition. This step can be carried out, for example, by aprocedure similar to that of step (S3) described above.

In step (S304), the signal correlated to turbidity is detected with therespective turbidity sensor in a plurality of detection regions. Thedetection of the signals correlated to turbidity may be carried out, forexample, by a procedure similar to the method described in step (S4),for example, may be carried out at the end point of the reaction orsequentially during the reaction.

In step (S305), it is determined whether an amplification product exists(positive) or does not exist (negative) in the vicinity of each of thedetection regions based on the result of the detection. This step can becarried out, for example, by a procedure similar to that of step (S5)described above. In this embodiment, the “vicinity” is defined, forexample, as a region on the reaction field, located within such adistance from an edge of a plurality of detection regions is in a rangeof 0 to 10 mm.

In step (S306), the target nucleic acid is quantified from the number ofpositive detection regions, and/or the rise time of the signalscorrelated to turbidity. This step can be carried out, for example, bythe same procedure as that of step (S6) described above.

According to the method described above, the target nucleic acid can bequantified simply at high sensitivity.

Chip

According to a further embodiment, there is provided a chip to quantifyand detect a target nucleic acid in a sample. The chip includes asubstrate and a plurality of detection regions disposed in one space onone surface of the substrate. The distance between two adjacentdetection regions, that is, the closest interval between two detectionregions adjacent to each other from an end of one detection region to acorresponding end of the other detection region may be 0.1 mm or more, 1mm or more, or 0.1 mm to 10 mm. The interval should more preferably be 1mm to 10 mm, in which case, the detection accuracy can be furtherimproved.

In one embodiment of the chip, the substrate may comprises an electrodecorresponding to each of the detection regions as described above. Inanother embodiment, the substrate may comprises an optical sensorcorresponding to each of the detection regions as described above. Inyet another embodiment, the substrate may comprises a turbidity sensorcorresponding to each of the detection regions as described above.

The chip is a chip for detecting or quantifying target nucleic acid inthe following manner. That is, a reaction liquid containing a sample, aprimer set for isothermally amplifying the first sequence to obtain anamplification product, and amplification enzyme is placed on a substrateto form one reaction field which causes an amplification reaction. Then,the detection signal which varies with the increase of the amplificationproduct are detected in each of the detection regions,

each detection region is determined based on the result of the detectionas to whether an amplification product exists (positive) or anamplification product does not exist (negative) in its vicinity. Fromthe number of positive detection regions and/or the rise time of thedetection signal in each of the positive detection regions, the targetnucleic acid is detected or quantified.

The chip is a substrate in which any of these detection regionsdescribed above, for example, whose distance of two adjacent detectionregions may be 0.1 mm or more, 1 mm or more, 0.1 mm to 10 mm, or 1 mm to10 mm are disposed. For example, such substrates are substrates 1, 11,21, 31 or 41 shown in FIG. 2 or 3. The chip may further comprise a covermember which covers the surface of the substrate, where the detectionregions are disposed.

According to the chip described above, there is no need to dilute asample or divide the reaction liquid, but with such a simple process ofbringing the reaction liquid onto one continuous region on a substrate,the target nucleic acid can be detected at higher accuracy. For example,according to the chip of the embodiment, it is possible to detect, forexample, 1 to 10⁹ copies/mL of target nucleic acid contained in thesample.

Assay Kit

According to the further embodiment, there is provided an assay kit todetect and quantify a target nucleic acid in a sample. The assay kitcontains any one of the chips described above, a primer set forisothermally amplifying the first sequence to obtain an amplificationproduct and an amplification reagent.

As the primer set, any one of those primer sets described can be used,for example. The amplification reagent contains, for example,amplification enzyme, a substrate such as deoxynucleoside triphosphoricacid (dNTP), a thickener as a reaction reagent, a buffer for pHadjustment, a surfactant, ion for enhancing the annealing specificityand/or ion which gives rise to a cofactor of the amplification enzyme,reverse transcriptase, or a combination of any of these. As theingredients of these amplification reagents, any of those describedabove can be used.

The assay kit may further contain the first marker substance and/or thesecond marker substance described above.

According to the assay kit described above, there is no need to dilute asample or divide the reaction liquid, but with such a simple process ofbringing the reaction liquid onto one continuous region on a substrate,the target nucleic acid can be detected at higher accuracy. For example,according to the assay kit of the embodiment, it is possible to detect,for example, 1 to 10⁹ copies/mL of target nucleic acid contained in thesample.

Device for Detecting or Quantifying Nucleic Acid

FIG. 8 shows an example of a device for detecting or quantifying nucleicacid (the nucleic acid detecting or quantifying device). FIG. 8 is ablock diagram showing an example of a nucleic acid detecting orquantifying device 50. The nucleic acid detecting or quantifying device50 comprises a first memory 60, a processor 70, a second memory 80, anoutput unit 90, a measurement unit 100 and the like. The first memory60, processor 70, second memory 80, output unit 90 and measurement unit100 are electrically connected to each other via a bus 110.

The first memory 60 is, for example, a hard disk, solid state drive(SSD), flash memory or the like and constitutes a storage area with thesecond memory 80.

The first memory 60 stores various types of software or data. Thevarious types of software includes an operating system (OS), a datamanagement program, various types of application programs, etc. Thefirst memory 60 stores a program P.

The processor 70 executes the various types of software (programs)described above and controls the entire nucleic acid detecting orquantifying device 50. The processor 70 is, for example, a centralprocessing unit (CPU), micro processing unit (MPU), digital signalprocessor (DSP) or the like.

The processor 70 executes the program P which is, for example, stored inthe first memory 60 or read from the first memory 60 to the secondmemory 80, and thus functions as, for example, a measurement controller71 or a nucleic acid quantification unit 72.

The measurement controller 71 is a control means which controls devicesincluded in the measurement unit 100 (that is, for example, a chip 101,a measurement device 102, a liquid transfer device 103, a temperaturecontroller 104, etc., which will be described later) and storesmeasurement data D obtained from the measurement unit 100 in the firstmemory 60.

The nucleic acid quantification unit 72 is a quantification means whichquantifies a target nucleic acid in a sample based on measurement data Dstored in the first memory 60. Further, the nucleic acid quantificationunit 72 stores in the first memory 60 quantification data R whichindicates a result of the quantification of the target nucleic acid inthe sample.

The output controller 73 transmits the quantification data R stored inthe first memory 60 to the output unit 90. Note that the outputcontroller 73 may be included in the measurement controller 71 and/orthe nucleic acid quantification unit 72.

The measurement data D and/or the quantification data R may be stored inthe second memory 80.

The second memory 80 as the main memory is, for example, a random accessmemory (RAM) and is used as a work area or the like. The work area isused when the processor 70 executes various types of software.

The output unit 90 outputs quantification data R under the control ofthe output controller 73. The output unit 90 may be, for example, adisplay or printer.

The first memory 60 may be divided into two or more. The second memory80 may be divided into two or more. Or the first memory 60 and thesecond memory 80 may be handled as one memory. The first memory 60,processor 70, second memory 80, output unit 90 and bus 110 may be builtin an information processing device.

The measurement unit 100 includes the chip 101, measurement device 102,liquid transfer device 103 and temperature controller 104.

The chip 101 is disposed on the nucleic acid detecting or quantifyingdevice 50 so as to be detachable therefrom. The chip 101 comprises asubstrate 1 (see FIG. 2, part (a)) and a cover member (not shown) fixedto the substrate 1. The substrate 1 comprises, on one surface thereof, aplurality of detection regions each comprising, for example, a sensor 4(see to FIG. 2, part (a)). The sensor 4 is, for example, an electrodewhen using an electric signal for detection of nucleic acid, or anoptical sensor when using an optical signal, or a turbidity sensor whenusing a signal correlated to turbidity.

The following explanation is directed to an example case where thesubstrate 1 and the sensors 4 are contained in the chip 101 of themeasurement unit 100. Alternatively, as shown in part (b) of FIG. 2 orparts (a) to (c) of FIG. 3, the substrate 11, 21, 31 or 41 comprisingthe sensors 16, 26, 36 or 46, respectively, or the like may be used inreplace of the substrate 1 comprising the sensors 4.

A reaction field is formed as bringing the reaction liquid into thespace by the substrate 1 and the cover member, and a predeterminedamplification reaction occurs in the reaction field. The amplificationreaction is, for example, the isothermal amplification reactiondescribed above, or the like. An upper surface of the cover member isprovided with an inlet for introducing a reaction liquid and an outletfor extracting the air and/or discharging the reaction liquid.

The measurement device 102 is electrically connected to the sensors 4included in the chip 101, and is a measurement means to receivedetection signals transmitted from the sensors 4. When the sensors 4 areeach an electrode, the measurement device 102 can apply voltage to theelectrode of the sensor 4 to receive the detection signal transmittedfrom the sensor 4. The measurement device 102 produces the measurementdata D based on the detection signal received under the control of themeasurement controller 71. The measurement data D include, for example,digital values and the like, indicating the presence/absence, intensityand/or detection time of the detection signals obtained in the sensor 4.

The liquid transfer device 103 is a liquid transfer means to send and/orextract the reaction liquid to/from the chip 101. The liquid transferdevice 103 comprises, for example, an interface 103 a with the chip 101and a container 103 b to contain a liquid such as a reaction liquid. Theliquid transfer device 103 sends the liquid in the container 103 b intothe chip 101 via the interface 103 a under the control of themeasurement controller 71 in accordance with necessity.

Note that the nucleic acid detecting or quantifying device 50 may notnecessarily comprise a liquid transfer device 103. In this case, thereaction liquid is transferred to or extracted from the chip 101 whilethe chip 101 is removed from the nucleic acid detecting or quantifyingdevice 50 by a liquid transfer device separate from the nucleic aciddetecting or quantifying device 50.

The temperature controller 104 is a temperature controlling means tocontrol the temperature of the chip 101 under the control of themeasurement controller 71. The temperature controller 104 may comprise,for example, a heater or a Peltier element.

Note that the nucleic acid detecting or quantifying device 50 may notnecessarily comprise a temperature controller 104. In this case, thetemperature of the chip 101 may be controlled by a temperaturecontroller separate from the nucleic acid detecting or quantifyingdevice 50.

Further, at least one of the processor 70, first memory 60, secondmemory 80 and output units 90 may be included in the measurement unit100. In this case, at least one of the processor 70, first memory 60,second memory 80 and output units 90 should preferably be contained inthe measurement device 102.

The nucleic acid detection by the nucleic acid detecting or quantifyingdevice 50 described above can be carried out as follows, for example, bythe procedure shown in FIG. 9. FIG. 9 is a flowchart showing an exampleof the nucleic acid detection treatment using the nucleic acid detectingor quantifying device 50.

The operator loads the chip 101 on the measurement unit 100 in advanceby insertion or the like. Further, let us suppose that the container 103b of the liquid transfer device 103 is filled with a reaction liquid inadvance. Then, for example, with the operation by the operator, thenucleic acid detection quantification treatment by the nucleic aciddetecting or quantifying device 50 is started. In the nucleic aciddetection quantification treatment, the measurement controller 71executes the treatments of steps (S401) to (S404), the nucleic acidquantification unit 72 executes the treatment of step (S405), and theoutput controller 73 executes the treatment of step (S406).

In step (S401), the liquid transfer device 103 sends the reaction liquidin the container 103 b, under the control of the measurement controller71, onto the surface of the substrate 1 of the chip 101, where aplurality of detection regions are disposed.

In step (S402), the temperature controller 104 adjusts the temperatureof the reaction field under the control of the measurement controller71. When the reaction field is maintained at the isothermalamplification condition, the isothermal amplification reaction isstarted in the reaction field.

In step (S403), the measurement device 102 receives a detection signaltransmitted from the sensor 4 under the control of the measurementcontroller 71.

In step (S404), the measurement controller 71 produces measurement dataD from the detection signal received by the measurement device 102 andstores the measurement data D thus obtained in the first memory 60.

In step (S405), the nucleic acid quantification unit 72 reads themeasurement data D from the first memory 60, and quantifies the targetnucleic acid in the sample based on the measurement data D thus read.

Details of the treatment of the nucleic acid quantification unit 72 willbe described later with reference to FIG. 10. Note that the measurementdata D may be stored in the second memory 80. Further, the nucleic acidquantification unit 72 stores the quantification data R indicating theresult of the quantification of the target nucleic acid in the firstmemory 60.

In step (S406), the output controller 73 reads quantification data Rfrom the first memory 60, and outputs it via the output unit 90. Morespecifically, the quantification data R may be output, for example, to adisplay or a printer.

FIG. 10 is a flowchart showing an example of treatment of the nucleicacid quantification unit 72. FIG. 10 corresponds to the treatment of thestep (S405) of FIG. 9 executed by the nucleic acid quantification unit72.

The nucleic acid quantification unit 72 executes the followingtreatments of steps (S501) to (S503).

In step (S501), the quantification means of the nucleic acidquantification unit 72 reads the measurement data D stored in the firstmemory 60 or the second memory 80 in step (S404).

In step (S502), the nucleic acid quantification unit 72 detects datacontained in the measurement data D, that is, for example, temporalincrease/decrease and/or rise time of the signal value received from thesensor 4, and thus determines whether an amplification product exists ina vicinity (positive) or an amplification product does not exists in avicinity (negative) in each detection region.

In step (S503), the nucleic acid quantification unit 72 totals thenumber of positive detection regions and the number of negativedetection regions per chip.

Next, the nucleic acid quantification unit 72 calculates, in three steps(α) to (γ) described below, the quantity of the target nucleic acidpresent in the sample based on the number of the detection regionsexhibiting to be positive.

In step (α), if it is judged that the number of positive detectionregions is 0 (step (S504)), the nucleic acid quantification unit 72determines that no target nucleic acid exists in the sample (step(S505)).

In step (β), if it is judged that both of positive detection regions andnegative detection regions exist (step (S506)), the nucleic acidquantification unit 72 calculates the quantity of the target nucleicacid present in the sample from the number of the positive detectionregions, for example, by the above-described statistical method (step(S507)).

In process (γ), if it is judged that all the detection regions exhibitto be positive (step (S508)), the nucleic acid quantification unit 72compares the quantity of standard sample nucleic acid and apredetermined calibration curve of the rise time of the signal asdescribed above, to calculate out the quantity of the target nucleicacid present in the sample (step (S509)).

Further, in step (510), the nucleic acid quantification unit 72 storesthe quantification data R indicating the quantification result of thetarget nucleic acid obtained by the above-described steps (α) to (γ) inthe first memory 60.

The nucleic acid detecting or quantifying device according to theembodiment can detect or quantify target nucleic acid in a simple mannerwith higher accuracy than the conventional techniques. Further,according to the nucleic acid detecting or quantifying device, it ispossible to conduct the examination of the target nucleic acid in ashorter time than that of the conventional techniques.

EXAMPLES Example 1

The time variations of the rise times of the current and potential inreaction liquids containing different numbers of copies of amplificationproduct were examined using substrates comprising an array electrode.

Manufacture of Substrate

A flow channel having a width and a height (=1 mm×1 mm) was provided ona glass surface of Pyrex (registered trademark) (d=0.8 mm), and thus asubstrate was formed. Then, thin films of titanium (500 nm) and gold(2,000 nm) were formed on a bottom of the flow channel and thenselectively etched using a pattern of resist AZP4620 as a mask. Thus,eight gold/titanium electrodes (p=200 μm) (working electrodes) wereformed. For every two active electrodes, a reference electrode and acounter electrode were formed to corresponding thereto.

Preparation of Reaction Liquid

Reaction liquids were prepared, which respectively contain 0 copy, 10²copies, 10³ copies, 10⁴ copies and 10⁵ copies and 106 copies of anartificial sequence (1 μL) of parvovirus (shown in TABLE 1 as SEQ ID NO:1), F3 primer (SEQ ID NO: 2), B3 primer (SEQ ID NO: 3), FIP primer (SEQID NO: 4), BIP primer (SEQ ID NO: 5) and Lb primer (SEQ ID NO: 6) as aLAMP primer shown in TABLE 2, RuHex (25 μM), KCl (60 mM), magnesium ion(8 mM), ammonium ion (10 mM), betaine (0.8 M), dNTPs (1.4 mM each) andpolymerase (GspSSD) (8 units).

TABLE 1 VP gene of Parvo virus (SEQ ID NO: 1)AAACGCTAATACGACTCACTATAGGGCGATCTACGGGTACTTTCAATAATCAGACGGAATTTAAATTTTTGGAAAACGGATGGGTGGAAATCACAGCAAACTCAAGGAGACTTGTACATTTAAATATCCCAGAAAGTGAAAATTATAGAAGAGTGGTTGTAAATAATTTGGATAAAAGTGCAGTTAACGGAAACATGGCTTTAGATGATACTCATGCACAAATTGTAACACCTTGGTCATTGGTTGATGCAAATGCTTGGGGAGTTTGGTTTAATCCAGGAGATTGGCAACTAATTGTTAATACTATGAGTGAGTTGCATTTAGTTAGTTTTGAACAAGAAATTTTAATGTTGTTTTAAAGACTGTTTCAGAATCTGGTACTCAGCCACCAACTAAAGTTTATAATAATGATTTAACTGCATCATTGATGGTTGCATTAGATAGTAATAATACTATGCCATTTACTCCAGCAGCTATGAGATCTGAGACATTGGGTTTTTATCCATGGAAACCAACCATACCAACTCCATGGAGATATTATTTTCAATGGGATAGAACATTAATACCATCTCATACTGGAACTAGTGGCACACCAACAAATATATACCATGGTACAGATCCAGATGATGTTCAATTTTATACTATTGAAAATTCTGTGCCAGTACACTTACTAAGAACAGGTGATGAATTTGCTACAGGAACATTTTTTTTTGATTGTAAACCATGTAGACTAACACATACATGGCAAACAAATAGAGCATTGGGCTTACCACCATTTCTAAATTCTTTGCCTCAAGCTGAAGGAGGTACTAACTTTGGTTATATAGGAGTTCAACAAGATAAAAGACGTGGTGTAACTCAAATGGGAAATACAAACTATACTGAAGCTACTATTATGAGACCAGCTGAGGTTGGTTATAGTGCACCATATTATTCTTTTGAGGCGTCTACACAAGGGCCATTTAAAACACCCTTCCCTTTAGTGAGGGTTAATAA

TABLE 2 SEQ ID NO Sequence 2 F3 GAGATATTATTTTCAATGGGATAGAAC 3 B3CAATGCTCTATTTGTTTGCCATG 4 FIP GAACATCATCTGGATCTGTACCAACCATCTCATACTGGAACTAGTGGC 5 BIP CTGTGCCAGTACACTTACTAAGAGTGTTAGTCTACATGGT TTACAATC 6 LbACAGGTGATGAATTTGCTACAGG

LAMP Amplification Reaction

These reaction liquids were brought onto the surface of the substratecomprising electrodes, and they were warmed isothermally at 67° C., tostart the amplification reactions. As the amplification reactionsproceeded, the electric signals were measured by the LSV method (sweeprate: 0.5 V/s). A detection region with an electrode which detected asignal of 1 nA/min or higher was detected within 60 minutes after thereaction started was determined as “positive”, and a detection regionwith an electrode of less than 1 nA/min was determined as “negative”.The results are shown in FIG. 11. Part (a) of FIG. 11 shows therelationship between the initial number of templates and the positiverate, and part (b) shows the relationship between the initial number oftemplates and the increase time (rise time) of the current value.

Under the conditions of the initial number of templates of 10³ copies ormore, the positive rate was 100%. Further, on this condition, theinitial number of templates and the increase time of the current valuehave a linear relation with respect to each other. Here, the correlationcoefficient (R²) indicated 0.93 and the inclination indicated −2.2. Fromthis result, it was suggested that when the initial number of templateswas 10³ copies or more, the number of initial templates can bequantified from the current value.

On the other hand, under the conditions of the initial number oftemplates of 10² copies or less, negative detection regions existed, andthe positive rate was 88% for 102 copies and 25% for 10¹ copies. Fromthis result, when less than 10³ copies, it was suggested that theinitial number of templates can be quantified from the positive rate.

Example 2

Using the results of Example 1, a calculation table was created toestimate the concentration of the target nucleic acid from the positiverate for the case where the target nucleic acid of less than 10³ copiesin a sample was to be quantified using the substrate of Example 1.

Calculation of Reaction Volume Per Electrode

In order to estimate the concentration of target nucleic acid from thepositive rate, the most probable number method which uses the formulabelow was used to estimate a most probable number (estimated quantity oftarget nucleic acid: MPN).MPN=1/m×2.303×log((number of detection regions)/(number ofnegatives))  Formula 3where m is a reaction volume (μL) per electrode.

The reaction volume, which is a parameter necessary to calculate themost probable number was calculated from the result of Example 1.

When Formula 3 is transformed, we obtainm−2.303×log((total number of electrodes)/(number of negatives))/MPN.When m is obtained from the result of 10² copies/50 μL of Example 1,i.e., 2 copies/μL, it was 1.03 μL. When m is obtained from the result of10¹ copies/50 μL of Example 1, i.e., 0.2 copies/μL, it was 1.44 μL. Anaverage of these two values is taken, and m=1.2 μL was used for thecalculation table. The most probable number was calculated using an MPNcalculator.

Similarly, a substrate comprising 80 electrodes was examined and acalculation table was created.

The calculation table thus created is shown in TABLE 3.

TABLE 3 Calculation table for quantification of target nucleic acidusing MPN method 95%-reliable zone Positive rate Estimated Conc.*Copies/μL) (%) (Copies/μL) n = 8 n = 80 100 — >1.7 >3.7 87.5 1.7 0.7-4.21.3-2.3 75 1.2 0.5-2.8 0.9-1.5 62.5 0.8 0.3-2.0 0.6-1.1 50 0.6 0.2-1.60.4-0.8 37.5 0.4 0.1-1.2 0.3-0.6 25 0.2 0.1-1.0 0.2-0.4 12.5 0.1 0.0-0.80.1-0.2 0 — <0.1 <0.0 *Estimated concentration (/μL) = 1/1.2 μL × 2.303× log ((number of electrodes(n))/(number of negatives))

It has been clarified from the above-described tests that according tothe method of the embodiment, a low-concentration target nucleic acidcan be quantified from the positive rate without preparing a calibrationcurve. Further, with an increased number of electrodes, the accuracy ofthe quantification of a low-concentration target nucleic acid can begreatly improved. Moreover, when the sample is not diluted, the MPNmethod can be adapted for the quantification of low-concentration targetnucleic acids, whereas middle- to high-concentration (10² copies/500 μL)target nucleic acids can be quantified from the rise time of the signalby preparing a calibration curve in advance.

Example 3

The optimal distance between electrodes was examined using the substrateof Example 1.

The flow channel had a width×height=1 mm×1 mm, and therefore it wasconsidered from the reaction volume calculated in Example 2 that theelectrodes can detect signals from the region in which the distance fromthe edge of the electrode is within about 0.6 mm. In order to increasethe degree of integration, the interval between electrodes shouldpreferably be shorter, but if excessively short, an amplificationproduct produced from one target nucleic acid may be detected by aplurality of electrodes. Thus, it has been clarified that in order toavoid this, the distance from one end of an electrode to an end of anadjacent electrode thereto should necessarily be at least 1 mm or more.

While certain embodiments have been described, these embodiments havebeen presented by way of example only, and are not intended to limit thescope of the inventions. Indeed, the novel embodiments described hereinmay be embodied in a variety of other forms; furthermore, variousomissions, substitutions and changes in the form of the embodimentsdescribed herein may be made without departing from the spirit of theinventions. The accompanying claims and their equivalents are intendedto cover such forms or modifications as would fall within the scope andspirit of the inventions.

What is claimed is:
 1. A method of quantifying one kind of targetnucleic acid containing a first sequence in a sample, the methodcomprising: preparing a substrate on which a plurality of detectionregions are arranged, without attaching any nucleic acid probe on thedetection regions, wherein a surface of the substrate on which thedetection regions are arranged is not divided for the detection regions;forming one reaction field by placing, on the surface of the substrate,one reaction liquid containing the sample, a primer set for isothermallyamplifying the first sequence to obtain a first amplification product,and an amplification enzyme; maintaining the reaction field in anisothermal amplification condition; detecting a detection signal varyingwith an increase of the first amplification product in each of theplurality of detection regions; determining, for each of the pluralityof detection regions, whether the first amplification product exists inits vicinity (positive) or the first amplification product does notexist in its vicinity (negative) based on the result of the detecting;and detecting or quantifying the target nucleic acid in the one reactionliquid based on the number of positive detection regions and on a risetime of the detection signal in each of the positive detection regions.2. The method of claim 1, wherein the isothermal amplification conditionis a LAMP reaction condition.
 3. The method of claim 1, wherein thesubstrate comprises an electrode corresponding to each of the detectionregions, the reaction liquid further contains a first marker substanceproducing the electric signal which varies with the increase of thefirst amplification product, and the detection signal is an electricsignal obtained by oxidation or reduction reaction of the first markersubstance.
 4. The method of claim 3, wherein the first marker substanceis ruthenium hexaamine.
 5. The method of claim 1, wherein the substratecomprises an optical sensor corresponding to each of the detectionregions, the reaction liquid further contains a second marker substanceproducing an optical signal which varies with the increase of the firstamplification product, and the detection signal is an optical signal. 6.The method of claim 1, wherein the substrate comprises a turbiditysensor corresponding to each of the detection regions, and the detectionsignal is a signal correlated to a turbidity of the reaction liquid. 7.The method of claim 1, wherein a distance between each adjacent pair ofthe plurality of detection regions is 1 mm or more.
 8. A chip fordetecting or quantifying one kind of target nucleic acid containing afirst sequence in one sample, the chip comprising: a substrate, and aplurality detection regions disposed in one space on one surface of thesubstrate without attaching any nucleic acid probe on the detectionregions, a distance between each adjacent pair of the plurality ofdetection regions being 1 mm to 10 mm, wherein the one surface of thesubstrate is not divided for the detection regions; one reaction liquidcontaining the sample, a primer set for isothermally amplifying thefirst sequence to obtain a first amplification product, andamplification enzyme is placed on the substrate to form one reactionfield which causes an amplification reaction, the detection signal whichvaries with the increase of the first amplification product is detectedin each of the detection regions, each detection region is determinedbased on the result of the detection as to whether the firstamplification product exists in its vicinity (positive) or the firstamplification product does not exist in its vicinity (negative), and thetarget nucleic acid in the one reaction liquid is detected or quantifiedfrom the number of positive detection regions and/or the rise time ofthe detection signal in each of the positive detection regions.
 9. Thechip of claim 8, wherein the substrate comprises an electrodecorresponding to each of the detection regions.
 10. The chip of claim 8,wherein the substrate comprises an optical sensor corresponding to eachof the detection regions.
 11. The chip of claim 8, wherein the substratecomprises a turbidity sensor corresponding to each of the detectionregions.
 12. An assay kit comprising: a chip of claim 8, a primer setfor isothermally amplifying the first sequence to acquire the firstamplification product and an amplification reagent.
 13. The assay kit ofclaim 12, further comprising: a first marker substance producing anelectric signal which varies with the increase of the firstamplification product.
 14. The method of claim 1, which quantifies onlyone kind of target nucleic acid.